Type 1 ryanodine receptor (RyR1) is an intracellular ligand-gated Ca2+ release channel in skeletal muscle where it is responsible for the increase of free cytoplasmic Ca2+ concentration leading to muscle contraction. Defects in this key protein or in modulation of its activity cause aberrant Ca2+ mobilization from the sarcoplasmic reticulum resulting in several muscle disorders such as Malignant Hyperthermia, Central Core and Multi-minicore Diseases. Lack of high-resolution structure of RyR1 currently limits our ability to understand how this channel functions both in native and disease states. The structural analysis of RyRs is exceptionally challenging due to their large size (~2.3 MDa), location in the membrane environment and dynamic nature. To date, single particle cryo-EM is the most viable methodology for structural analysis of such large integral membrane proteins. However, cryo-EM studies of RyRs confront an additional hurdle due to the presence of detergent in cryospecimen. This raises a number of issues with optimization of ice thickness and EM imaging to produce images with a good contrast for reliable 3D reconstruction. The reduced image contrast is the main impediment to producing high-resolution structures of membrane proteins in general. Achieving a high- resolution structure of RyR1 by cryo-EM clearly requires a breakthrough in methodology for cryo-specimen preparation and imaging conditions. In this project, we aim to develop a method for vitrification of RyR1 for single particle cryo-EM analysis that utilizes the use of a new class of surfactants, amphipathic polymers, in place of detergent to keep the channel protein soluble and in its functional form in aqueous detergent-free solution (aim1). By using this approach we anticipate to circumvent detergent-imposed difficulties in cryo-EM studies and to achieve the 3D reconstruction of RyR1 in a closed state at subnanometer resolution. We then propose to reconstitute RyR1 channel into small unilamellar vesicles and to use a variant of single particle reconstruction to solve the channel structure in the lipid membrane (aim 2). New computational tools will be developed for this project within the framework of EMAN/EMAN2 software in order to achieve the structure of intact RyR1 at resolutions beyond the current ~1 nm and to establish its 3D architecture in membrane environment. The determined structures will reveal mechanistically informative protein features that will allow important insights into RyR1 channel function. Once optimal methodologies are established, we anticipate to extend the structural analysis of RyR1 to near-atomic resolution and to different physiologically relevant functional states. The methods developed, as part of this research, will have broad applicability to studies of other ion channels and large membrane protein complexes in near-native state.